PROJECT SUMMARY Ductal carcinoma in situ (DCIS) is the most common form of pre-invasive breast cancer, accounting for 30% of all diagnosed breast cancers. Clinical evident estimates that ~40% of patients with DCIS left untreated will progress to invasive breast cancer. Although, the mechanisms by which pre-invasive DCIS cells acquire the ability to invade the adjacent stroma are largely unknown. The long-term research goals of these studies are to understand the molecular switch that occurs when pre-invasive lesions break through the basement membrane to invade the surrounding stroma. The proposed studies aim to dissect the role of macrophages in premalignancy and to identify the mechanisms that promotes tumor invasion into the stroma. Using mouse models of early progression, we have shown that macrophages are recruited to pre-invasive lesions and polarized toward a tumorigenic phenotype, and secret the cytokine Gas6. Our hypothesis is that macrophages exert distinct functions at different stages of premalignancy, and macrophage-derived Gas6 promotes the transition to invasive cancer. This hypothesis will be tested by the following aims: Aim 1: To characterize macrophage phenotypes in pre-invasive lesions and determine whether their function is dependent on Gas6. Aim 2: To determine the contribution of stromal-derived Gas6 on the progression of early stage lesions. Our studies will utilize several mouse models, a unique 3-D culture system, and single cell RNA-seq to dissect the role of macrophage-derived Gas6 in the progression of premalignancy. By understanding the mechanisms of invasion, this study could identify biomarkers to be used in therapeutics to predict patient outcome and direct treatment. The results of this study will significantly enhance our understanding of DCIS biology and early stage breast cancer progression. Through the course of this fellowship I will learn indispensable techniques, enhance my writing and presentation skills, and fine tune my critical thinking skills. I have formed a strong mentoring committee to help fill in the gaps in my scientific career. I will receive training in mouse models, breast cancer biology, human and mouse pathology, and bioinformatics, all of which will prepare me for the next stage of my career. I have access to a highly collaborative, breast cancer-rich research environment, comprised of basic scientists, clinicians, translational scientists, and epidemiologist. There are excellent core facilities from multiple universities and institutions within close proximity. There are many opportunities to participate in professional development workshops, as well as attend outstanding seminars and scientific retreats. My sponsor, co-sponsors and I have devised specific career development sessions that will enhance my training, facilitate new collaborations, and play an integral role in developing into a leading, independent researcher.